Liquid chromatography component

ABSTRACT

The present invention aims to provide a liquid chromatography component including a column and a prefilter, which is hard to cause an increase of supplied liquid pressure even when the measurement of a sample is repeated. The present invention is a liquid chromatography component, which includes: a column with filler particles filled therein; and a prefilter, the filler particles having an average particle size in the range of 2 to 20 μm, the prefilter having a filtering particle size in the range of ⅙ to ⅓ of the average particle size of the filler particles.

TECHNICAL FIELD

The present invention relates to a liquid chromatography componentincluding: a column with filler particles filled therein; and aprefilter, wherein the component is hard to cause an increase in thesupplied liquid pressure even when the measurement of a sample isrepeated. The present invention also relates to a liquid chromatographycomponent including: a column with filler particles filled therein; anda prefilter, wherein the prefilter requires replacement at almost thesame frequency as the column does. Further, the present inventionrelates to a liquid chromatography component including: a column withfiller particles filled therein; and a prefilter, wherein the column andthe prefilter are integrated with each other, require no complicatedreplacement operation, and show excellent separation performance.

BACKGROUND ART

In the fields of organic chemistry, biochemistry, medicine, and thelike, liquid chromatography has been widely used for measurement oranalysis of a component in a sample. In the medical field, for example,liquid chromatography has been employed for measuring hemoglobin A1c,which is an indicator for diabetes diagnosis. Hemoglobin A1c isglycosylated hemoglobin that has blood sugar chemically bound to anN-terminus of a β chain of hemoglobin. A proportion of hemoglobin A1C inhemoglobins, that is, a proportion of glycosylated hemoglobin in a sumof glycosylated hemoglobin and non-glycosylated hemoglobin is consideredto reflect an average blood sugar level in a period of one to twomonths. Therefore, a hemoglobin A1c value (%) which represents theproportion of hemoglobin A1c in hemoglobins has been widely used as anindicator for diabetes diagnosis because the value does not showtemporary fluctuation unlike a blood sugar level.

Filters such as an in-line filter for filtering foreign substances arelocated in a flow channel between a liquid chromatographysample-injection device to a column. Such filters are disposed in orderto prevent foreign substances from clogging the channel, particularlythe column body, and thereby changing the pressure of a supplied liquid.Particularly a prefilter, which is located on the upstream side of thecolumn to filter foreign substrates, is an important filter linkingdirectly to the column clogging caused by the foreign substances.

Examples of the foreign substances, which are captured on the filters,include: those contained in a mobile phase, a reaction reagent, and thelike; those from a part of an analyzer, such as a feed pump; and thosederived from a sample. These foreign substances are adsorbed on thesurface of filler particles filled in the column or to a detector cell,and as a result, may adversely affect the measurement or analysis.Accordingly, there have been developed various filters for efficientlycapturing these foreign substances.

However, when the filtration efficiency of the filters is increased inorder to more efficiently capture the foreign substances, the foreignsubstances may more easily clog the filters to cause the change in thesupplied liquid pressure.

Particularly when a large number of samples are continuously measured asin analysis of hemoglobins or when a sample high in foreign substanceslike a hemolyzed blood sample is measured, the foreign substances clogthe filter and often cause an increase in the supplied liquid pressure.The change in the supplied liquid pressure may lead to a failure inaccurate and quick measurement or analysis of the sample.

Examples of a common method of suppressing the increase in the suppliedliquid pressure, caused by clogging of the filter, include a method ofincreasing a filtration area of the filter, a method of increasing aporosity of the filter, and a method of modifying a configuration of thefilter. However, when the filtration area or the porosity of the filteris increased too much, the sample or the mobile phase in the filter maybe diffused too much, which reduces the accuracy of the measurement oranalysis.

As an example of modifying a configuration of the filter, PatentDocument 1 discloses a method of using a two-layer filter composed oflayers with different pore sizes. Patent Document 2 discloses a methodof using a two-layer structure composed of a filter paper sheet and afilter. These methods are designed to prevent the clogging attributed tothe foreign substances by a combination use of the two different filterswithout decreasing the filtration efficiency for the foreign substances.However, these ideas have been established by taking only the filterinto consideration, and no studies have been made covering the columnwhere the clogging attributed to the foreign substances poses a problemas in the filter.

For example, a column for analysis of hemoglobins, which is used formeasuring a hemoglobin A1c value, is usually replaced each time afterthe measurement of 1500 to 3000 samples is completed, although dependingon the number of samples guaranteed by a manufacturer. In contrast tothis, the prefilter, which is located on the upstream side of thecolumn, is usually replaced each time after the measurement of hundredsof samples is completed. Thus, the filters require the replacement moreoften than the column does, which is a significant burden in terms ofoperation and cost.

-   Patent Document 1: Japanese Kokai Publication No. Hei-02-262054(JP-A    Hei-02-262054)-   Patent Document 2: Japanese Kokai Publication No. Hei-05-203634(JP-A    Hei-05-203634)

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

The present invention aims to provide a liquid chromatography componentincluding: a column with filler particles filled therein; and aprefilter, wherein the component is hard to cause an increase insupplied liquid pressure even if the measurement of a sample isrepeated, and wherein the prefilter requires replacement at almost thesame frequency as the column does. Further, the present inventionrelates to a liquid chromatography component including: a column withfiller particles filled therein; and a prefilter, wherein the column andthe prefilter are integrated with each other, require no complicatedreplacement operation, and show excellent separation performance.

The present invention is a liquid chromatography component, whichincludes: a column; and a prefilter, the filler particles having anaverage particle size in the range of 2 to 20 μm, the prefilter having afiltering particle size in the range of ⅙ to ⅓ of the average particlesize of the filler particles.

The present invention is described in detail below.

The liquid chromatography component of the present invention includes acolumn and a prefilter.

In the column, the filler particles are filled in a cylindricalcontainer.

Examples of the filler particles include: inorganic particles such assilica; organic particles comprising resins such as a styrene-divinylbenzene copolymer; and these particles with a surface having anion-exchange group bonded thereto.

The lower limit of the average particle size of the filler particles is2 μm, and the upper limit thereof is 20 μm. If the average particle sizeof the filler particles is smaller than 2 μm, the supplied liquidpressure in the column may become too high, and a burden on the analyzermay increase too much. If the average particle size of the fillerparticles is larger than 20 μm, the separation performance is degraded,and for example, if the particles with such an average particle size areused in measurement of hemoglobin A1c, hemoglobins may be insufficientlyseparated. The lower limit of the average particle size of the fillerparticles is preferably 6 μm, and the upper limit thereof is preferably12 μm.

The average particle size of the filler particles can be measured with alaser diffraction-type particle size distribution measuring apparatus.

The lower limit of the inner diameter of the column is preferably 2.0ram, and the upper limit thereof is preferably 6.0 mm. If the innerdiameter of the column is smaller than 2.0 mm, the linear velocity of amobile phase flowing inside the column may become too high, and thesupplied liquid pressure may increase too much. If the inner diameter ofthe column is larger than 6.0 mm, the sample or the mobile phase insidethe column may be diffused too much, possibly resulting in deteriorationof the separation performance. The lower limit of the inner diameter ofthe column is more preferably 3.0 mm, and the upper limit thereof ismore preferably 5.0 mm.

The lower limit of the length of the column is preferably 10 mm, and theupper limit thereof is preferably 50 mm. If the column is shorter than10 mm, the separation performance may be degraded along with a decreasein the number of theoretical plates. If the column is longer than 50 mm,elution of a sample may require a longer time to extend the measurementtime, or the supplied liquid pressure may increase. The lower limit ofthe length of the column is more preferably 15 mm, and the upper limitthereof is more preferably 40 mm.

It is preferable that a filter is disposed on each of the upstream anddownstream sides of the column, thereby preventing the filler particlesfilled in the column from leaking from a cylindrical container. Thesefilters are not disposed with the view of capturing foreign substances,and it is sufficient if the filters can prevent the leakage of thefiller particles from the cylindrical container. When thebelow-mentioned prefilter is integrated with the column and locatedextremely close thereto, the filter on the upstream side may not bedisposed, and instead, the prefilter may prevent the filler particlesfilled in the column from leaking from the cylindrical container.

The prefilter may be made of paper, resin, metal, and the like.Particularly preferably used is a stainless-steel filter having athree-layer structure composed of layers with different pore sizes,disclosed in Japanese Kokai Publication No. 2006-189427 (JP-A2006-189427).

The filtering surface of the prefilter may have a circular shape, or anyother shape.

The prefilter has a filtering particle size in the range of ⅙ to ⅓ ofthe average particle size of the filler particles. Foreign substancesthat are not captured by the prefilter and pass therethrough are notcaptured in the column either to pass through spaces between the fillerparticles inside the column. As a result, clogging is suppressed in theprefilter and the column. If the filtering particle size of theprefilter is smaller than ⅙ of the average particle size of the fillerparticles, the clogging of the prefilter may occur much earlier thanthat of the column, so that the prefilter may require replacementfrequently. If the filtering particle size of the prefilter is largerthan ⅓ of the average particle size of the filler particles, theclogging of the prefilter may occur less often, but foreign substancesmay clog up spaces between the filler particles inside the column,possibly resulting in deterioration of the separation performance.Preferably, the filtering particle size is in the range of ⅕ to ⅓ of theaverage particle size of the filler particles.

The filtering particle size means a particle size at which, whenstandard particles with a known particle size are filtered through theprefilter, the capture rate of the standard particles is 95% or higher.As the standard particles, commercially available polystyrene standardparticles produced by Moritex Corp. can be used.

The capture rate (%) of the standard particles is measured in thefollowing procedures.

The prefilter is connected to a liquid chromatograph, and purified wateris allowed to flow as a mobile phase. The standard particle sample isallowed to flow at a general feed speed, for example, 1.7 mL/min. Then,the peak area (1) of the obtained chromatogram is calculated. The peakarea (1) reflects the amount of the standard particles having passedthrough the prefilter without being captured thereby.

Next, the prefilter is replaced with a pipe, and then the same standardparticle sample is allowed to follow. Then, the peak area (2) of theobtained chromatogram is calculated. The peak area (2) reflects theamount of the standard particle having being supplied.

Based on the peak area (1) and the peak area (2), the capture rate (%)of the standard particles is calculated from the following formula.

Capture rate (%) of standard particles=100−(peak area (1)/peak area(2))×100

The lower limit of the effective filtration area of the prefilter ispreferably 7 mm², and the upper limit thereof is preferably 80 mm². Ifthe effective filtration area of the prefilter is smaller than 7 mm²,the clogging of the prefilter may occur more often because the rangewhere the foreign substances can be captured is narrow. If the effectivefiltration area of the prefilter is larger than 80 mm², the sample orthe mobile phase inside the prefilter may be diffused too much, possiblyresulting in degradation of the separation performance. The lower limitof the effective filtration area of the prefilter is more preferably 12mm², and the upper limit thereof is more preferably 65 mm².

The lower limit of the thickness of the prefilter is preferably 0.1 mm,and the upper limit thereof is preferably 10 mm. If the thickness of theprefilter is smaller than 0.1 mm, the clogging of the prefilter mayoccur more often. If the thickness of the prefilter is larger than 10mm, the sample or the mobile phase inside the prefilter may be diffusedtoo much, possibly resulting in deterioration of the separationperformance. The lower limit of the thickness of the prefilter is morepreferably 0.2 mm, and the upper limit thereof is more preferably 3 mm.

The lower limit of the porosity of the prefilter is preferably 60%. Ifthe porosity of the prefilter is smaller than 60%, the clogging of theprefilter may occur more often. The lower limit of the porosity of theprefilter is more preferably 65%. The upper limit of the porosity of theprefilter is preferably 90% since a prefilter with an excessively highporosity possibly does not satisfy the desired filtering particle size.

In the liquid chromatography component of the present invention, thecolumn and the prefilter may be disposed independently, or may beintegrally disposed within a single cylindrical container. In each case,the component is jointed to a pipe of the analyzer such that theprefilter is located on the upstream side of the column.

When being disposed independently, the column and the prefilter can bereplaced independently. When being integrally disposed, the column andthe prefilter can be replaced together, so that the replacementoperation becomes easier. Further, the integrated column-prefilteroccupies less space, so that the analyzer can be downsized. Further,when the column and the prefilter are integrally disposed, the distancebetween the two is substantially zero, so that the sample and the mobilephase is hardly diffused between the column and the prefilter. As aresult, the separation performance is improved, and the measurement timecan be more shortened.

The cylindrical container comprises a material with a proper strength,and can accommodate the filler particles, or both of the fillerparticles and the prefilter. Examples of the material for thecylindrical container include metals such as stainless steel andtitanium, resins such as fluorine resin and polyether ether ketone, andglass materials.

The cylindrical container may be a single-piece one or a disassemblableone.

It is preferable that the prefilter or the cylindrical container issubjected to a surface treatment, thereby preventing non-specificadsorption to the surface. The surface treatment means a chemical and/ora physical treatment that is provided on a surface in order to modifythe surface properties. Specific examples of the surface treatmentinclude: surface modification by a thermal or acid oxidation reaction;and a blocking treatment of covering the surface with a substance havingdesired characteristics, such as a hydrophilic substance and ahydrophobic substance. Proteins such as bovine serum albumin, globulin,lactoferrin, and skim milk; silicone; and fluorine resins can be used asthe substance used in the blocking treatment.

According to the present invention, the life of the prefilter becomesvery long by adjustment of a combination of the average particle size ofthe filler particles filled in the column and the filtering particlesize of the prefilter. Therefore, the replacement frequency of thecolumn can be made equivalent to that of the prefilter.

Since the replacement frequency of the column is equivalent to that ofthe prefilter, the prefilter, which is located on the upstream side ofthe column, requires no replacement during a period of use of thecolumn. Further, the column and the prefilter are integrally disposed,so that the replacement operation of the two can be made easier. Also,since the integrated column-prefilter occupies less space, the analyzercan be downsized.

The present invention also includes a liquid chromatography component,wherein a column with filler particles filled therein and a prefilterbody are integrally disposed within a single cylindrical container.

For example, when the liquid chromatography component includes theprefilter and the column, the replacement frequency of the column can bemade equivalent to that of the prefilter.

The column and the prefilter, which require replacement at almost thesame frequency, are integrally disposed within a single cylindricalcontainer, whereby the column and the prefilter can be replaced in asingle replacement operation. Thus, the replacement operation can beefficiently carried out. In addition, the column and the prefilter aredisposed with substantially no distance therebetween, so that the sampleor the mobile phase is hardly diffused between the column and theprefilter. As a result, the separation performance is improved, and themeasurement time can be more shortened.

FIG. 1 shows one example of a liquid chromatography component of thepresent invention, in which the column and the prefilter areindependently disposed.

FIG. 2 shows one example of a liquid chromatography component of thepresent invention, in which the column and the prefilter are integrallydisposed within a single cylindrical container.

EFFECT OF THE INVENTION

The present invention provides a liquid chromatography componentincluding a column and a prefilter, wherein the component is hard tocause an increase in supplied liquid pressure even if the measurement ofa sample is repeated, and wherein the prefilter requires replacement atalmost the same frequency as the column does. The present invention alsoprovides a liquid chromatography component including a column and aprefilter, wherein the column and the prefilter are integrated with eachother, require no complicated replacement operation, and show excellentseparation performance.

BEST MODE FOR CARRYING OUT THE INVENTION

The aspects of the present invention are described below in more detailbased on examples. The present invention is not limited to the examples.

Example 1 (1) Preparation of Prefilter

A sintered stainless-steel fiber filter sheet with a thickness of 0.4 mmand a porosity of 70% was punched into a circular shape with a diameterof 9.0 mm to give a sintered stainless-steel fiber filter with afiltration area of 63.59 mm². The sintered stainless-steel fiber filtersheet had a three-layer structure composed of: a filter layer (outerlayer 1) with a pore diameter of 12 μm and a thickness of 0.1 mm; afilter layer (inner layer) with a pore diameter of 3 μm and a thicknessof 0.2 mm; and a filter layer (outer layer 2) with a pore diameter of 12μm and a thickness of 0.1 mm, stacked in this order. The obtained filterwas subjected to a blocking treatment with bovine serum albumin, therebypreventing non-specific adsorption of hemoglobins.

The surface-treated filter was secured with a polytetrafluoroethylenepacking and then housed in a polyether ether ketone holder with a threadportion that can be connected to a channel. Thus, a prefilter wasprepared. The prefilter had an effective filtration area, which is anarea except for the packing-contacting area, of 50.24 mm².

The obtained prefilter was measured for filtering particle size andfound to be 3 μm.

(2) Preparation of Column

A mixture containing tetraethylene glycol dimethacrylate (product ofShin-Nakamura Chemical Co., Ltd.) 300 g, triethylene glycoldimethacrylate (product of Shin-Nakamura Chemical Co., Ltd.) 100 g, andbenzoyl peroxide (product of Kishida Chemical Co., Ltd.) 1.0 g was addedto a 3% aqueous solution of polyvinyl alcohol (product of NipponSynthetic Chemical Industry Co., Ltd.). Under stirring, the resultingmixture in a reaction vessel was polymerized under nitrogen atmosphereat 80° C. for one hour.

Next, as a monomer containing an ion-exchange group,2-methacrylamide-2-methylpropanesulfonic acid (product of TOAGOSEI CO.,LTD.) 100 g, polyethylene glycol methacrylate (product NOF CORPORATION,ethylene glycol chain n=4) 100 g were dissolved in ion-exchanged water.This mixture was further added into the above-mentioned reaction vesselafter one hour-polymerization, and then polymerized at 80° C. for 2hours under stirring and nitrogen atmosphere. The obtained polymercomposition was washed with water and acetone, thereby yieldingion-exchange group-containing particles.

The obtained particles 10 g were immersed in an ozone water 300 mL witha dissolved ozone gas concentration of 100 ppm and then stirred for 30minutes. After completion of the stirring, the mixture was centrifugedwith a centrifuge (Himac CR20G, produced by Hitachi, Ltd.), therebyremoving supernatant fluid. This process was repeated twice to obtainfiller particles.

The average particle size and the CV value of the obtained fillerparticles were measured with the laser diffraction-type particle sizedistribution measuring apparatus, and were found to be values of 10 μmand 14%, respectively.

The obtained filler particles were charged into a cylindrical containerwith 4.6 mm in inner diameter and 20 mm in length. Thus, a column wasprepared. A filter (the reference number 6 in FIGS. 1 and 2) with 6.5 mmin diameter, prepared by punching of the above-mentioned sinteredstainless-steel fiber filter, was disposed on each of the upstream anddownstream sides of the column, so as to prevent the filler particlesfrom leaking from the cylindrical container.

(3) Production of Liquid Chromatography Component

The obtained prefilter and column were disposed as illustrated in FIG. 1to produce a column/prefilter discrete-type liquid chromatographycomponent. Hereafter, the liquid chromatography component having theconfiguration of FIG. 1 is referred to as a discrete-type liquidchromatography component.

The obtained prefilter and column were disposed as illustrated in FIG. 2to produce a column/prefilter integrated-type liquid chromatographycomponent. Hereafter, the liquid chromatography component having theconfiguration of FIG. 2 is referred to as an integrated-type liquidchromatography component.

Examples 2 to 6, Comparative Examples 1 and 2

Discrete-type liquid chromatography components (FIG. 1) andintegrated-type liquid chromatography components (FIG. 2) were producedin the same manner as in Example 1, except that the prefilters and thecolumns shown in Table 1 were used.

The effective filtration area of the prefilter was adjusted by a changein the diameter of the punching mold. The filtering particle size of theprefilter was adjusted by a change in the pore diameter of the innerlayer of the filter. The average particle size of the filler particleswas adjusted by a change in the rotation speed of the stirring upon thepolymerization.

TABLE 1 Comparative Comparative Example 1 Example 2 Example 3 Example 4Example 5 Example 6 Example 1 Example 2 Prefilter Effective filtrationarea 50.24 50.24 50.24 23.75 50.24 50.24 50.24 50.24 (mm²) Thickness(mm) 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 Porosity (%) 70 70 70 70 70 70 7070 Filtering particle size (μm) 3.0 2.5 1.8 3.0 2.0 4.0 5.0 1.4 ColumnAverage particle size 10.0 10.0 10.0 10.0 6.0 12.0 10.0 10.0 of fillerparticles (μm) Inner diameter of 4.6 4.6 4.6 4.6 4.6 4.6 4.6 4.6 column(mm) Length of column (mm) 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0

(Evaluation) (1) Evaluation of Supplied Liquid Pressure

Any of the discrete-type liquid chromatography component produced inExamples and Comparative Examples to the following system was jointed tothe following systems, and thereby setting up a liquid chromatographyanalyzer.

Feed pump: LC-20AD (produced by Shimadzu Corp.)Auto sampler: SIL-20AC (produced by Shimadzu Corp.)Detector: SPD-M20A (produced by Shimadzu Corp.)Column oven: CTO-20AC (produced by Shimadzu Corp.)

A total of 3000 samples were successively measured with this liquidchromatography analyzer under the following analysis conditions.

Eluent:

first solution 50 mmol/L phosphate buffer (pH 5.3)

second solution 250 mmol/L phosphate buffer (pH 8.0) containing 0.05% byweight of polyoxyethylene (20) sorbitan monolaurate (produced by WakoPure Chemical Industries, Ltd.)

Measuring time: 50 secondsFlow rate: 1.7 mL/minColumn temperature: 40° C.Detection wavelength: 415 nmSample: whole blood from a healthy human was diluted 201-fold with ahemolysing agent (phosphate buffer (pH 7.0) containing 0.1% by weight ofpolyoxyethylene (10) octylphenyl ether (produced by Wako Pure ChemicalIndustries, Ltd.)) to yield a sample to be evaluated.Amount of sample injection: 10 μL

After completion of the continuous analysis of the 3000 samples, thepressure when the first solution of the eluent was supplied into each ofthe column and the prefilter was measured. The pressure increase in thefirst solution was calculated from the following formula. When thepressure increase in both of the column and the prefilter is 0.3 MPa orlower, the case is evaluated as “O”. When the pressure increase ineither the column or the prefilter exceeds 0.3 MPa, the case isevaluated as “x”.

Table 2 shows the results.

Pressure increase=(Pressure of supplied liquid after continuous analysisof 3000 samples)−(Pressure of supplied liquid before sample analysis)

TABLE 2 Pressure increase Pressure increase in column (MPa) in prefilter(MPa) Evaluation Example 1 0 0 ◯ Example 2 0 0.1 ◯ Example 3 0 0.3 ◯Example 4 0 0 ◯ Example 5 0 0.2 ◯ Example 6 0 0 ◯ Comparative 0.6 0 XExample 1 Comparative 0 0.8 X Example 2

Table 2 shows that when the liquid chromatography components of Examples1 to 6 were used, the pressure increase in each of the prefilter and thecolumn was 0.3 MPa or lower in every case. Even after the continuousanalysis of the 3000 samples, the pressure was hardly changed in each ofthe column and the prefilter. In Examples 2, 3, and 6, the pressure ofthe first solution in the prefilter was slightly increased but ininsignificant range.

In contrast to this, the pressure increase was observed in the columnwhen the liquid chromatography component of Comparative Example 1 wasused. The reason for this is considered that the combination of thecolumn and the prefilter was not suitable and that foreign substancesthat had passed through the prefilter without being captured clogged upspaces of the filler particles inside the column during the repeatedmeasurement. When the liquid chromatography component of ComparativeExample 2 was used, an increase in the pressure of the first solutionwas observed in the prefilter. The reason for this is considered thatexcessive foreign substances were captured by the prefilter, and thecaptured substances caused clogging of the prefilter during the repeatedmeasurement.

(2) Durability Evaluation

The discrete-type or integrated-type liquid chromatography componentproduced in Example 4 was jointed to the following systems, therebysetting up a liquid chromatography analyzer.

Feed pump: LC-20AD (produced by Shimadzu Corp.)Auto sampler: SIL-20AC (produced by Shimadzu Corp.)Detector: SPD-M20A (produced by Shimadzu Corp.)Column oven: CTO-20AC (produced by Shimadzu Corp.)

With these liquid chromatography analyzers, the hemoglobin A1c value wascontinuously measured under the following analysis conditions, and thedurability was evaluated.

Table 3 shows the results (HbA1c value). Eluent:

first solution 50 mmol/L phosphate buffer (pH 5.3)second solution 250 mmol/L phosphate buffer (pH 8.0)containing 0.05% by weight of polyoxyethylene (20) sorbitan monolaurate(produced by Wako Pure Chemical Industries, Ltd.).Measuring time: 50 secondsFlow rate: 1.7 mL/minColumn temperature: 40° C.Detection wavelength: 415 nmSample to be loaded: whole blood from a healthy human was diluted201-fold with a hemolyzing agent (phosphate buffer (pH 7.0) containing0.1% by weight of polyoxyethylene (10) octylphenyl ether (produced byWako Pure Chemical Industries, Ltd.)) to prepare a sample to be loaded.Sample to be evaluated: each of glyco Hb control levels 1 and 2(produced by Sysmex International Reagents Co., Ltd.) was dissolved inan water for injection 200 μL, and then diluted 101-fold with aphosphate buffer (pH 7.0) containing 0.1% by weight of polyoxyethylene(10) octylphenyl ether (produced by Wako Pure Chemical Industries,Ltd.)) to prepare a sample to be evaluated.

After the measurement of every 200 samples to be loaded, 3 samples to beevaluated were measured, and average values thereof were used for theevaluation.

Amount of sample injection: 10 μL

TABLE 3 Column/prefilter Column/prefilter discrete-type integrated-typeThe number Hemoglobin A1c value (%) Hemoglobin A1c value (%) of sampleLevel 1 Level 2 Level 1 Level 2 0 5.2 10.2 5.2 10.2 200 5.2 10.2 5.210.2 400 5.2 10.3 5.2 10.2 600 5.3 10.2 5.2 10.2 800 5.2 10.2 5.2 10.21000 5.2 10.2 5.2 10.2 1200 5.3 10.2 5.3 10.3 1400 5.2 10.2 5.2 10.21600 5.2 10.3 5.2 10.2 1800 5.2 10.2 5.2 10.2 2000 5.2 10.1 5.2 10.12200 5.2 10.2 5.2 10.2 2400 5.2 10.2 5.2 10.2 2600 5.3 10.2 5.3 10.22800 5.2 10.3 5.2 10.3 3000 5.2 10.2 5.2 10.2 Avg. 5.22 10.21 5.21 10.21SD 0.04 0.05 0.03 0.04 CV 0.77 0.49 0.66 0.43

As shown in Table 3, each of the discrete-type and the integrated-typeliquid chromatography components in Example 4 exhibited excellentdurability.

INDUSTRIAL APPLICABILITY

The present invention can provide the liquid chromatography componentcomposed of a column and a prefilter, wherein the component is hard tocause an increase in supplied liquid pressure even when the measurementof a sample is repeated, and wherein the prefilter requires replacementat almost the same frequency as the column does.

The present invention can provide the liquid chromatography componentincluding a column and a prefilter, wherein the component is hard tocause an increase in supplied liquid pressure even when the measurementof a sample is repeated, and wherein the prefilter requires replacementat almost the same frequency as the column does.

The present invention also provides the liquid chromatography componentincluding a column and a prefilter, wherein the column and the prefilterare integrated with each other, require no complicated replacementoperation, and show excellent separation performance.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view showing an example of a liquid chromatographycomponent of the present invention, wherein a column and a prefilter areindependently disposed.

FIG. 2 is a schematic view showing an example of a liquid chromatographycomponent of the present invention, wherein a column and a prefilter areintegrally disposed.

EXPLANATION OF SYMBOLS

-   1 Discrete-type liquid chromatography component (Column part)-   1′ Discrete-type liquid chromatography component (Prefilter part)-   2 Integrated-type liquid chromatography component-   3 Joint pipe-   4 Part filled with filler particles in column-   5 Polytetrafluoroethylene packing-   6 Filter for preventing leakage of filler particles-   7 Prefilter-   8 Polyether ether ketone holder

1-3. (canceled)
 4. A method for assaying blood samples comprising,applying a blood sample to a liquid chromatography component whichcomprises: a column with filler particles filled therein; and aprefilter; said filler particles having an average particle size in therange of 2 to 20 μm, and said prefilter having a filtering particle sizein the range of ⅙ to ⅓ of the average particle size of said fillerparticles, wherein the blood sample is assayed.
 5. The method accordingto claim 4, wherein the column and the prefilter are integrally disposedat a substantially zero distance.